Dissecting the role of one neuronal RhoGEF amongst many: the Kalirin-7 null mouse

描述（申请人提供）：Rho GTPases在神经元发育和树突棘的形成和功能中起关键作用。智力迟钝与个体Rho蛋白、激活Rho蛋白的鸟嘌呤核苷酸交换因子（GEFs）以及激活Rho蛋白的下游靶标的缺陷有关。Kalirin（一种大的双Rho GEF蛋白）的表达缺陷与阿尔茨海默病脑iNOS水平升高和精神分裂症患者死后前额叶皮质脊柱密度降低有关。此外，Kalirin与DISC1（一种候选精神分裂症基因）和HAP1（一种亨廷顿蛋白相互作用基因）相互作用。单核苷酸多态性预测了Kalirin功能的大量杂合个体，只有一个表达拷贝或正常拷贝和突变拷贝。这些观察结果为分析小鼠提供了一个令人信服的案例，其中Kalirin的表达可以在发育期间和成年期间被操纵。哺乳动物也表达Trio，这是一种高度同源但非冗余的基因。单Kalirin/Trio基因在果蝇和秀丽隐杆线虫的神经系统内外发挥着重要作用。根据我们对培养神经元的研究，Kalirin在轴突的起始和生长以及树突的生长中起着核心作用。过度表达Kalirin的主要成人剪接变体Kalirin-7，会增加锥体神经元和正常分枝的中间神经元中树突棘的形成。Kalirin-7和Kalirin-7（由不同启动子产生的n端截断变体）表达的减少导致脊柱形成和维持的缺陷。我们建立了可以改变Kalirin-7和Kalirin-7表达的小鼠模型。缺乏kal7in -7/ kal7in -7 （Kal7KO）特有的单外显子的小鼠出生频率只有预期的一半，但能存活到成年并繁殖。该外显子（Kal7+/KO）杂合的小鼠Kalirin-7和Kalirin-7水平降低，并表现出突触传递缺陷。在超微结构水平上，Kal7KO小鼠的正常兴奋性突触数量减少，加上许多正常小鼠未见的异常突触谱。当在高零迷宫中进行测试时，Kal7+/KO和Kal7KO小鼠表现出焦虑样行为的逐步减少。Kal7外显子被lox-p位点包围的小鼠（Kal7CKO）允许组织特异性的、发育调节的Kalirin-7/ Kalirin-7的消除。这些小鼠将通过行为测试、突触前和突触后元素的形态学评估、切片的电生理记录和亚细胞部分的生化分析进行评估。通过表达外源性Kalirin-7可以挽救Kal7KO小鼠制备的海马神经元的脊柱形成，从而可以详细分析Kalirin-7和分离的Sec14p、spectrin-like、DH和PH结构域的作用。将评估Kal7在响应Shank3、GluR2和Neuroligin-1等蛋白的脊柱形成中的作用。六种已知的人类kal7in -7突变体挽救脊柱形成和突触功能的能力将通过Kal7KO小鼠进行评估。公共卫生相关性：树突棘上的兴奋性突触负责神经元之间的大部分交流。树突棘的数量和形状对发育线索、环境刺激和激素变化作出反应。脊柱形态的改变在学习和记忆中起着关键作用，很明显，许多信号通路影响脊柱的形成和功能。Kalirin-7是Rho家族的小GTP结合蛋白的激活剂，定位于树突棘，是已知能够增加树突棘数量的少数因素之一。我们计划使用我们产生的Kalirin-7敲除小鼠来阐明控制脊柱形成和结构的途径。

DESCRIPTION (provided by applicant): Rho GTPases play key roles in neuronal development and in the formation and function of dendritic spines. Mental retardation is associated with deficits in individual Rho proteins, in the guanine nucleotide exchange factors (GEFs) that activate Rho proteins and in downstream targets of activated Rho proteins. Deficits in expression of Kalirin, a large, dual Rho GEF protein, are associated with increased iNOS levels in Alzheimer's disease brain and with decreased spine density in post-mortem prefrontal cortex from schizophrenic patients. In addition, Kalirin interacts with DISC1 (a candidate schizophrenia gene) and HAP1 (a Huntingtin interactor). Single nucleotide polymorphisms predict a significant number of individuals heterozygous for Kalirin function, with only one expressed copy or a normal and a mutated copy. These observations make a compelling case for analyzing mice in which expression of Kalirin can be manipulated both during development and in the adult. Mammals also express Trio, a highly homologous, but non- redundant gene. The single Kalirin/Trio gene in Drosophila and C. elegans plays an essential role within and outside of the nervous system. Based on our studies in cultured neurons, Kalirin plays a central role in axon initiation and outgrowth and in dendritic growth. Over-expression of the major adult splice variant of Kalirin, Kalirin-7, increases the formation of dendritic spines in pyramidal neurons and in normally aspiny interneurons. Reductions in the expression of Kalirin-7 and Kalirin-7 (an N-terminally truncated variant generated from a different promoter) result in deficits in spine formation and maintenance. We generated mouse models in which expression of Kalirin-7 and Kalirin-7 can be varied. Mice lacking the single exon unique to Kalirin-7/ Kalirin-7 (Kal7KO) are born at half the expected frequency, but survive to adulthood and reproduce. Mice heterozygous for this exon (Kal7+/KO) have diminished levels of Kalirin-7 and Kalirin-7 and show deficits in synaptic transmission. At the ultrastructural level, Kal7KO mice have a reduced number of normal excitatory synapses, plus many aberrant synaptic profiles not seen in normal mice. When tested in the elevated zero maze, Kal7+/KO and Kal7KO mice show a graded decrease in anxiety-like behavior. Mice in which the Kal7 exon is surrounded by lox-p sites (Kal7CKO) allow tissue-specific, developmentally regulated elimination of Kalirin-7/ Kalirin-7. These mice will be assessed using behavioral tests, morphological assessment of pre- and post-synaptic elements, electrophysiological recordings of slices and biochemical analysis of subcellular fractions. Spine formation in hippocampal neurons prepared from Kal7KO mice can be rescued by expressing exogenous Kalirin-7, allowing detailed analysis of the role of Kalirin-7 and the isolated Sec14p, spectrin-like, DH and PH domains. The role of Kal7 in spine formation in response to proteins such as Shank3, GluR2 and Neuroligin-1 will be assessed. The ability of the six known human Kalirin-7 mutants to rescue spine formation and synaptic function will be assessed using the Kal7KO mice. PUBLIC HEALTH RELEVANCE: Excitatory synapses onto dendritic spines account for much of the communication that goes on between neurons. The number and shape of dendritic spines respond to developmental cues, environmental stimuli and hormonal changes. Changes in spine morphology play key roles in learning and memory and it is clear that many signaling pathways affect spine formation and function. Kalirin-7, an activator of small GTP binding proteins of the Rho family, is localized to dendritic spines and is one of a small number of factors known to be capable of increasing the number of dendritic spines. We plan to use the Kalirin-7 knockout mouse that we generated to elucidate the pathway(s) controlling spine formation and structure.